Prostate cancer is a malignant disease that is prevalent in Western countries, and it is also becoming remarkably prevalent in Japan (Non-patent Document 1). The number of deaths from prostate cancer in Japan in 2009 exceeded 10,000 to reach 10,036, and is still increasing (Non-patent Document 2). Therefore, there is also a social demand for improvement of the diagnostic technique and development of a new therapeutic method for prostate cancer.
The prostate-specific antigen (PSA), which was separated from seminal plasma in 1979 by Wang et al., is a glycoprotein having a molecular weight of 34 kDa composed of 237 amino acids, and is a substance identical to human kallikrein 3 (Non-patent Document 3). Measurement of serum PSA is currently widely used not only for diagnosis and judgment of recurrence of prostate cancer, but also for prognostication, and judgment of therapeutic effects. Although serum PSA has good detection sensitivity for prostate cancer, its specificity for detection of cancer is low, since it is a prostate-specific antigen rather than a prostate cancer-specific antigen and PSA may also increase in cases of a benign disease such as benign prostatic hyperplasia or prostatitis, or rectal examination or ejaculation. Therefore, in cases where the reference value 4 ng/mL is used as the serum PSA cutoff in work-up, the false-positive rate is high, and this results in unnecessary prostatic biopsy which imposes physical burden on the patient. It is actually said that, in the so-called gray zone, where the serum PSA value is 4 to 10 ng/mL, the positive rate of prostate cancer is 25 to 30%, and that unnecessary biopsy is carried out in about 70% of the cases within this range (Non-patent Document 4). Thus, a new biomarker that allows non-cancer patients to avoid unnecessary biopsy and enables more efficient detection of prostate cancer patients than serum PSA is being strongly demanded.
Improvement of the method of collection of clinical samples from patients is also demanded. Although blood is a common clinical sample, the amount of blood that can be sampled may be limited in cases where blood samples need to be frequently collected in monitoring of disease over time. Use of a clinical sample whose collection is less burdensome for patients is more preferred. Urine is excreted every day and can be noninvasively collected, and urinary markers are ideal materials for examination also from an ethical point of view. Moreover, since the duct of the prostate opens at the urethra, various substances directly secreted from the prostate can be detected by comparing spot urine with post-prostatic massage urine. Actually, known diagnostic methods using a urine sample include (1) urinary RNAs, (2) urinary DNAs, (3) urinary proteins or (4) urinary metabolites. Among these, the prostate cancer gene 3 (PCA3), which encodes a non-coding RNA, has been reported to show high expression in prostate cancer but low expression in a normal prostate (Non-patent Document 5), and some diagnostic methods for prostate cancer by detection of urinary PCA3 have been reported in recent years (Non-patent Document 6). However, at clinical sites, such methods are still inferior to examination of serum PSA, and diagnostic methods using other urinary components are still being studied. Thus, establishment of a novel method for prostate disease testing using a urinary peptide or protein fragment associated with the disease is of high clinical significance.
Urine contains various peptides and protein fragments having molecular weights of not more than 10 kDa, and the amount of these peptides and fragments excreted reaches several ten milligrams per day even in healthy individuals (Non-patent Document 7). Urine can be noninvasively sampled, has only low activity of protease, and contains substances derived from blood, kidney, bladder, ovary, prostate and the like. Therefore, multifaceted proteomics studies targeting proteins having molecular weights of not less than 10 kDa are being carried out in search of disease-associated markers (Non-patent Documents 8 and 9).
Examples of the common method for comprehensive analysis of peptides and proteins include two-dimensional electrophoresis and liquid chromatography. However, such methods are suitable for molecular fractions corresponding to molecular weights of not less than 10,000, and they are not suitable for specific detection of low-molecular-weight proteins and peptides. A protein chip technology by the combination of Surface Enhanced Laser Desorption/Ionization (SELDI) and Time-of-flight/Mass spectrometry: (TOF/MS) was developed and utilized for detection of novel tumor markers and the like (Non-patent Document 10). However, since structure determination (amino acid sequence determination) cannot be done with disease-associated marker peaks detected by SELDI-TOF/MS, the method is not suitable for confirmatory clinical tests.
In terms of prostate cancer, Theodorescu et al. performed capillary electrophoresis (CE)-TOF/MS analysis of urine samples, and 12 peptide peaks were detected and identified. These peaks were reported as candidates for prostate cancer-associated markers (Non-patent Document 11), but they have not been practically used yet. Okamoto et al. used urine voided after prostatic massage as samples to perform SELDI-TOF/MS analysis within the mass range of m/z 2,500 to 150,000 using sinapic acid as a matrix, and detected 72 peaks significantly different from the peaks observed for benign prostatic hyperplasia (Non-patent Document 12). However, since most of the detected peaks have not been identified, they have not been clinically applied.
Patent Documents 1 to 8, which aim at industrial application, disclose short PSA fragments of not more than about 20 to 30 residues. All of these are peptides each composed of a region that induces a specific biological effect such as an epitope, identified by analysis of the PSA sequence, and are not natural products but artificial products. There is no disclosed information on the fact that urine actually contains such short PSA fragments and that such fragments can be used as markers for prostate cancer testing, and none of these fragments has been practically used for clinical tests.